To characterize the tumor microenvironment after standard radiation therapy (SRT) and pulsed radiation therapy (PRT) in Lewis lung carcinoma (LLC) allografts. Subcutaneous LLC tumors were established in C57BL/6 mice. Standard RT or PRT was given at 2Gy/d for a total dose of 20Gy using a 5days on, 2days off schedule to mimic clinical delivery. Radiation-induced tumor microenvironment changes were examined after treatment using flow cytometry and antibody-specific histopathology. Normal tissue effects were measured using noninvasive (18)F-fluorodeoxyglucose positron emission tomography/computed tomography after naïve animals were given whole-lung irradiation to 40Gy in 4weeks using the same 2-Gy/d regimens. Over the 2weeks of therapy, PRT was more effective than SRT at reducing tumor growth rate (0.31±0.02mm(3)/d and 0.55±0.04mm(3)/d, respectively; P<.007). Histopathology showed a significant comparative reduction in the levels of Ki-67 (14.5%±3%), hypoxia (10%±3.5%), vascular endothelial growth factor (2.3%±1%), and stromal-derived factor-1α (2.5%±1.4%), as well as a concomitant decrease in CD45(+) bone marrow-derived cell (BMDC) migration (7.8%±2.2%) after PRT. The addition of AMD3100 also decreased CD45(+) BMDC migration in treated tumors (0.6%±0.1%). Higher vessel density was observed in treated tumors. No differences were observed in normal lung tissue after PRT or SRT. Pulsed RT-treated tumors exhibited slower growth and reduced hypoxia. Pulsed RT eliminated initiation of supportive mechanisms utilized by tumors in low oxygen microenvironments, including angiogenesis and recruitment of BMDCs.